WO2023004415A2

WO2023004415A2 - Sars-cov-2 vaccine using bacterial spores expressing antigenic fragments

Info

Publication number: WO2023004415A2
Application number: PCT/US2022/074048
Authority: WO
Inventors: Mehdi MIRSAEIDI; Abdolrazagh Hashemi SHAHRAKI; Mohammad VAHED
Original assignee: University Of Miami
Priority date: 2021-07-22
Filing date: 2022-07-22
Publication date: 2023-01-26
Also published as: WO2023004415A3

Abstract

Disclosed are compositions comprising a SARS-CoV-2 vaccine that can be administered orally. The vaccine can be on a platform comprising a spore coat protein from a spore forming bacteria, such as Bacillus subtilis. Also disclosed are methods of preventing or treating a COVID-19 infection comprising providing the SARS-CoV-2 vaccine to a subject.

Description

SARS-COV-2 VACCINE USING BACTERIAL SPORES EXPRESSING

ANTIGENIC FRAGMENTS

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Serial No. 63/224,638, filed July 22, 2021, the disclosure of which is expressly incorporated herein by reference.

FIELD

The present disclosure relates to SARS-COV-2 vaccines using bacterial spores.

BACKGROUND

Genetic engineering techniques provide the ability to use microorganisms as bioreactors and cell factories in industrial processes as well as food fermentation. Bacillus subtilis ( . subtilis ), due to its safety profile, is a natural candidate in the food and pharmaceutical industries (Fazelnia 2021; Won 2020; Yu 2019; Ayala 2017).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of deaths in the world so far (Nasiri 2020). The virus has been mutating to improve its ability to evade immune systems. The mutations in the spike-protein also increased the virus infectivity. Currently, several novel variants of SARS-CoV-2 circulate in the community, but the most impactful ones are B.1.351 (South Africa) and P.1 (Brazilian and Japanese) variants, and B.1.617 (Indian or Delta, and Delta-Plus) (Elena Quinonez 2021).

Animal studies show that vaccination with SAR-CoV-2 epitope peptides leads to protective immunity (Dai 2021). The viral epitope is profiled to contain four proteins including envelop (E), membrane (M), nucleocapsid (N), and spike (S) that activate the host T and B cells with cross-reactivity with HLA receptors (Stervbo 2020). It has been shown that the development of rapid, strong, and multifaceted responses to viral epitopes via CD4+ and CD8+ T cells increases the ability of the immune system to clear the infection in the early stages. Production of artificial peptide epitopes of viruses is fast, structurally stable, and free of any viral or microbial contamination. The most important advantage of this group of vaccines is the elimination of unwanted areas that trigger harmfully immune systems.

B. subtilis spores have been used as vaccine delivery vehicle for mucosal immunity and promoting the increase of antibody responses after oral administration with epitope antigens either admixed or adsorbed on the spore surface (Ricca 2014; Sibley 2014; Wang 2014; de Souza 2014; Song 2012). Bacillus sw /A-based vaccine has been developed by Lee and coworkers for Rotavirus in 2010 (Lee 2010). They concluded that intranasal inoculation with B. subtilis spore-based rotavirus vaccines is effective in generating protective immunity against rotavirus challenge in mice. A few years later Zhao et al. developed an orally delivered influenza vaccine based on B. subtilis spore expressing M2 protein (Zhao 2014).

Many people experience mild to moderate side effects after injected COVID-19 vaccination including local injection pain, fever, fatigue and skin rash. Safe and effective vaccines that can provide an alternative to injected vaccines are urgently needed.

SUMMARY

Disclosed herein is a composition comprising an isolated spore coat protein, or a functional fragment thereof, wherein the spore coat protein is from a spore-forming bacteria conjugated to an antigenic fragment of SARS-CoV-2. The spore coat protein can be from Bacillus subtilis , and can be part of a whole, functional spore, or can be a fragment thereof.

Also disclosed are vaccines which utilize the compositions disclosed herein. These vaccines are particularly suitable for oral and intranasal administration.

Further disclosed is a method of immunizing a subject against SARS-CoV-2 infection, the method comprising administering to the subject a vaccine, wherein the vaccine comprises an isolated spore coat protein or a functional fragment thereof, wherein said spore coat protein is from a spore-forming bacteria, wherein the spore coat protein is conjugated to an antigenic fragment of SARS-CoV-2.

Additionally, disclosed are methods of making a vaccine for SARS-CoV-2, the method comprising isolating a spore coat protein or a functional fragment thereof, wherein said spore coat protein is from a spore-forming bacteria, the method further comprising conjugating an antigenic fragment of SARS-CoV-2 to the spore coat protein.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description illustrate the disclosed compositions and methods.

FIG. 1 shows the schematic representing spike immobilization at the surface of Bacillus subtilis spores. Bacillus spore is first killed and adsorbed with B.1.351 (South Africa) and P.l (Brazilian and Japanese) and Delta variants SARS-CoV-2 spike and then used for immunization.

FIG. 2 shows covalent immobilization of SARS-CoV-2 spike on the activated spore surface utilizing EDC/NHS. EDC reacts with a carboxyl group on the spore, forming an amine-reactive O-acylisourea intermediate. The addition of NHS stabilizes the amine- reactive intermediate via converting it to an amine-reactive NHS ester, therefore increasing the efficiency of EDC-mediated coupling reactions. This intermediate can react with an amine on the SARS-CoV-2 spike and a stable amide bond is formed between the carboxyl group of the spore and the amino group of the enzyme.

DETAILED DESCRIPTION

Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

Definitions

As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.

Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10”as well as “greater than or equal to 10” is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:

“Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

An “increase” can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant.

A “decrease” can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also, for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.

“Inhibit,” “inhibiting,” and “inhibition” mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic. It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to.

By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.

The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, avian, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

“Biocompatible” generally refers to a material and any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause significant adverse effects to the subject.

“Comprising” is intended to mean that the compositions, methods, etc. include the recited elements, but do not exclude others. “Consisting essentially of ’ when used to define compositions and methods, shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. “Consisting of ’ shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and/or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.

A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be “positive” or “negative.” For example, a negative control can be an untreated or mock treated control. A positive control can be a control with a known positive response.

“Effective amount” of an agent refers to a sufficient amount of an agent to provide a desired effect. The amount of agent that is “effective” will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like. Thus, it is not always possible to specify a quantified “effective amount.” However, an appropriate “effective amount” in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts. An “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

A “pharmaceutically acceptable” component can refer to a component that is not biologically or otherwise undesirable, e.g., the component may be incorporated into a pharmaceutical formulation provided by the disclosure and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When used in reference to administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

“Pharmaceutically acceptable carrier” (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use. The terms “carrier” or “pharmaceutically acceptable carrier” can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) and/or various types of wetting agents. As used herein, the term “carrier” encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein.

“Pharmacologically active” (or simply “active”), as in a “pharmacologically active” derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.

“Therapeutic agent” refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the term “therapeutic agent” is used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.

“Therapeutically effective amount” or “therapeutically effective dose” of a composition refers to an amount that is effective to achieve a desired therapeutic result. In some embodiments, a desired therapeutic result is the control of type I diabetes. In some embodiments, a desired therapeutic result is the control of obesity. Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as pain relief. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and/or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years.

An “immunogenic molecule” means a recombinant protein, native protein, or artificial small molecule that stimulates an immune response in a subject. Preferably, an immunogenic molecule does not adversely affect a subject when administered.

An “immune response” or “immunological response” means, but is not limited to, the development of a cellular and/or antibody-mediated immune response to the composition or vaccine of interest. Usually, an immune or immunological response includes, but is not limited to, one or more of the following effects: the production or activation of antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. Preferably, the subject will display either a therapeutic or a protective immunological (memory) response such that resistance to new infection will be enhanced and/or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction in number of symptoms, severity of symptoms, or the lack of one or more of the symptoms associated with the infection of a pathogen, and/or a delay in the of onset of symptoms.

Those of skill in the art will understand that the compositions disclosed herein may incorporate known injectable, physiologically acceptable sterile solutions. For preparing a ready-to-use solution for parenteral injection or infusion, aqueous isotonic solutions, e.g., saline or plasma protein solutions, are readily available. In addition, the compositions of the present invention can include diluents, isotonic agents, stabilizers, or adjuvants.

“Diluents”, as used herein, can include water, saline, dextrose, ethanol, glycerol, and the like. “Isotonic agents” can include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. “Stabilizers” include albumin and alkali salts of ethylenediaminetetraacetic acid, among others.

Herein, an “adjuvant” or “adjuvants” can include aluminum hydroxide and aluminum phosphate, saponins e.g., Quil A, QS-21 (Cambridge Biotech Inc., Cambridge Mass.), GPI- 0100 (Galenica Pharmaceuticals, Inc., Birmingham, Ala.), non-metabolizable oil, mineral and/or plant/vegetable and/or animal oils, polymers, carbomers, surfactants, natural organic compounds, plant extracts, carbohydrates, water-in-oil emulsion, oil-in-water emulsion, and water-in-oil-in-water emulsion. The emulsion can be based in particular on light liquid paraffin oil (European Pharmacopeia type); isoprenoid oil such as squalane or squalene; oil resulting from the oligomerization of alkenes, in particular of isobutene or decene; esters of acids or of alcohols containing a linear alkyl group, more particularly plant oils, ethyl oleate, propylene glycol di(caprylate/caprate), glyceryl tri-(caprylate/caprate) or propylene glycol di oleate; or esters of branched fatty acids or alcohols, in particular isostearic acid esters. The oil is used in combination with emulsifiers to form the emulsion. The emulsifiers are preferably nonionic surfactants, in particular esters of sorbitan, mannide (e.g., anhydromannitol oleate), glycol, polyglycerol, propylene glycol, and oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene- polyoxy ethylene copolymer blocks, in particular the Pluronic products, especially L121. (See Hunter et ah, The Theory and Practical Application of Adjuvants (Ed. Stewart-Tull, D. E. S.), John Wiley and Sons, NY, pp 51-94 (1995) and Todd et ah, Vaccine 15:564-570 (1997).

Compositions of the invention also can include one or more pharmaceutical- acceptable earners. Herein, “pharmaceutical-acceptable carrier” or “veterinary-acceptable carrier” include any and all solvents, dispersion media, coatings, stabilizing agents, growth media, dispersion media, cell culture media and cell culture constituents, coatings, adjuvants, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like.

“Isolated” means altered “by the hand of man” from its natural state, e.g., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.

Severe acute respiratory syndrome coronavirus 2 (“SARS-CoV-2”) is a type of human coronavirus. Representative examples of human coronavirus can also include, but are not limited to, human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV- OC43), human coronavirus HKU1 (HCoV-HKUl), Human coronavirus NL63 (HCoV- NL63), severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome-related coronavirus (MERS-CoV).

In some embodiments, the coronavirus infection can be caused by an avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), Porcine epidemic diarrhea virus (PEDV), HCoV-229E, HCoV-OC43, HCoV-HKUl, HCoV-NL63, SARS- CoV, SARS-CoV-2, or MERS-CoV.

As used herein, “COVID-19” refers to the infectious disease caused by SARS-CoV- 2 and characterized by, for example, fever, cough, respiratory symptoms, rhinorrhea, sore throat, malaise, headache, chills, repeated shaking with chills, diarrhea, new loss of smell or taste, muscle pain, or a combination thereof.

In some embodiments, the subject with a coronavirus exhibits one or more symptoms associated with mild COVID-19, moderate COVID-19, mild-to-moderate COVID-19, severe COVID-19 (e.g., critical COVID-19), or exhibits no symptoms associated with COVID-19 (asymptomatic). It should be understood that in reference to the treatment of patients with different COVID-19 disease severity, “asymptomatic” infection refers to patients diagnosed with COVID-19 by a standardized RT-PCR assay that do not present with fever, cough, respiratory symptoms, rhinorrhea, sore throat, malaise, headache, or muscle pain.

In some embodiments, the subject with a coronavirus exhibits one or more symptoms selected from dry cough, shortness of breath, and fever. In other embodiments, the subject exhibits no symptoms associated with COVID-19 but has been exposed to another subject known or suspected of having COVID-19.

The originally discovered strain of SARS-CoV-2 has SEQ ID NO: 5. SARS-CoV-2 includes numerous variants including Indian variants (B.1.617-1, SEQ ID No: 1, and B.1.617- 2, SEQ ID NO: 2), South African variants (B.1.351, SEQ ID NO: 3), and Japanese/Brazilian variants (P.1, SEQ ID NO: 4). Further variants include, but are not limited to, Omicron variants (e.g., BA.l, BA.2, BA.4, BA.5, BA.2.75, and BA.3), Alpha variants (e.g., B.l.1.7), Epsilon variants (e.g., B.1.427, B.1.429), B.1.616(c) variant, Eta variants (e.g., B.1.525), Theta variants (e.g., P.3), B.1.620 variant, B.1.617.3 variant, B.1.214.2 variant, A.23.1 variant, A.27 variant, A.28 variant, C.16 variant, B.1.351 variant, B.1.1.7 variant, Iota variant (e.g., B.1.526), B.1.526.1 variant, B.1.526.2 variant, Zeta variant ( e.g., P.2), B.1.1.519 variant, AV. l variant, AT.l variant, C.36 variant, P.l variant, B.1.621 variant, C.37 variant, AY.4.2 variant, B.1.1.318 variant, C.1.2 variant, B.1.351 variant, P. l variant, B.1.640 variant, XF variant, and XD variant. The spike mutations of these variants include, but are not limited to, L452X, F486V, R493Q, N501Y, D614G, P681H, E484K, L452R, V483A, H655Y, G669S, Q677H, E484Q, P681R, S477N, Q414K, N450K, ins214TDR, V367F, Q613H, A653V, H655Y, N501T, P384L, K417N, A701V, E516Q, S494P, T478K, N439K, N679K, ins679GIAL, K417T, R346K, L452Q, F490S, P618R, A222V, Y145H, N679K, Y449H, E484X, T478K, F490R, N394S, R346S, Y449N, and 137-145de.+

Omicron BA.4&BA.5 Apt 20202(SEQ ID NO: 7)

MF VFL VLLPL V S SQC VNLITRT QLPP S YTN SFTRGVYYPDK VFRS S VLHSTQDLFLPF F SN VTWFH AIH V S GTN GTKRFDNP VLPFNDGV YF AS TEK SNIIRGWIF GTTLD SKTQ SLLIVNNATNVVIKVCEF QF CNDPFLGVYYHKNNKSWMESEFRVY S S ANNCTFEY VSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIY SKHTPINLGRDLPQGF S ALEPLV DLPIGINITRF QTLL ALHRS YLTPGD S S SGWT AGAAAYYV GYLQPRTFLLK YNENGT ITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFDEVFN ATRFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYAD SF VIRGNE VSQI APGQTGNIAD YNYKLPDDFTGC VI AWN SNKLD SK VGGNYNYRY RLFRK SNLKPFERDI S TEI Y O AGNKPCN GY AGVN C YFPLO S Y GFRPT Y GY GHOP YR V VVL SFELLH AP AT VCGPKK S TNL VKNKC VNFNFN GLTGT GVLTE SNKKFLPF Q QF GRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPV AIHADQLTPTWRVYSTGSNVF QTRAGCLIGAEYVNN S YECDIPIGAGIC AS Y QTQTK SHRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDC TMYICGD STEC SNLLLQ Y GSF CTQLKRALTGI AVEQDKNTQEVF AQ VKQIYKTPPIK YFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQ KFN GLT VLPPLLTDEMI AQ YT S ALL AGTIT S GWTF GAGA ALQIPF AMQM A YRFN GI GVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLVK QL S SKF GAI S S VLNDIL SRLDPPE AE V QIDRLIT GRLQ SLQT Y VTQQLIR A AEIRA SAN LAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTA PAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNN

TVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKN

LNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCS

CGSCCKFDEDDSEPVLKGVKLHYTLEDYKDDDDK

Omicron BA.4 (SEQ ID NO: 8)

IDMF VFLVLLPLV S SQCVNLTTRTQLPPAYTN SFTRGVYYPDKVFRS S VLHSTQDLF

LPFF SNVTWFHyiHVSGTNGTKRFDNPVLPFNDGVYF ASTEKSNIIRGWIF GTTLDS

KTQ SLLIVNN ATN V VIK V CEF QF CNDPFLD V YYHKNNK S WME SEFRV Y S S ANNCT

FEYVSQPFLMDLEGKQGNFKNLREF VFKNIDGYFKIY SKHTPINIVRDLPQGF S ALEP

LVDLPIGINITRF QTLLALHRS YLTPGD S SSGWTAGAAAYYV GYLQPRTFLLKYNEN

GTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFDE

VFNATRFASVYAWNRKRISNCVADYSVLYNFAPFFTFKCYGVSPTKLNDLCFTNV

Y AD SF VIRGNEVRQI APGQTGNIAD YNYKLPDDFTGC VIAWN SNKLD SK VSGNYN

YLYRLFRKSNLKPFERDISTEIYOAGNKPCNGVAGFNCYFPLOSYGFRPTYGVGHO

P YRV VVL SFELLHAP AT VCGPKK S TNL VKNKC VNFNFN GLTGT GVLTE SNKKFLPF

QQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTE

VP VAIHADQLTPTWRVYSTGSNVF QTRAGCLIGAEYVNN S YECDIPIGAGIC AS Y QT

QTKSHRRARS V ASQ SII AYTMSLGAEN S VAY SNN SI AIPTNFTIS VTTEILP V SMTKT S

VDCTMYICGDSTECSNLLLQYGSFCTQLKRALTGIAVEQDKNTQEVFAQVKQIYKT

PPIK YF GGFNF SQILPDP SKP SKRSFIEDLLFNK VTL AD AGFIKQ Y GDCLGDI AARDLI

CAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRF

N GIGVT QNVL YEN QKLI AN QFN S AIGKIQD SL S S T AS ALGKLQD V VNHN AQ ALNTL

VKQLSSHFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRAS

ANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNF

TTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGI

VNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEV

AKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLK

GCCSCGSCCKFDEDDSEPVLKGVKLHYTLEDYKDDDDK

Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

Compositions and Methods

Compositions

Disclosed herein is a composition comprising an isolated spore coat protein, or a functional fragment thereof, wherein the spore coat protein is from a spore-forming bacteria conjugated to an antigenic fragment of SARS-CoV-2.

The antigenic fragment of SARS-CoV-2 can comprise any of the variants disclosed herein. Examples include, but are not limited to, the “wild type,” or originally discovered SARS-CoV-2 (SEQ ID NO: 5), the Indian variants (B.1.617-1, SEQ ID NO: 1, and B.1.617- 2, SEQ ID NO: 2) the South African variant (B.1.351, SEQ ID NO: 3) and the Japanese/Brazilian variant (P.1, SEQ ID NO: 4). Further examples include Omicron variants (e.g., BA.4, SEQ ID NO: 7 and BA.5, SEQ ID NO: 8). There are many other variants which are known, or will be known, which are also contemplated herein. In some embodiments, the antigenic fragment of SARS-CoV-2 can have a sequence identity 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homologous with a sequence identity of any of the variants disclosed herein, including SEQ ID NOS: 1-5, 7-8. In some embodiments, the antigenic fragment of SARS- CoV-2 can have a sequence identity from 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100% homologous with the sequence identity of any of the variants disclosed herein, including SEQ ID NOS: 1-5, 7-8. In further embodiments, the antigenic fragment of SARS- CoV-2 can have a sequence identity 60% or greater, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, or 95% or greater homologous with a sequence identity of any of the variants disclosed herein, including SEQ ID NOS: 1-5, 7-8. In certain embodiments, the antigenic fragment of SARS-CoV-2 can have a sequence identity from 80% to 82%, 82% to 84%, 84% to 86%, 86% to 88%, 88% to 90%, 90% to 92%, 92% to 94%, 94% to 96%, 96% to 98%, or 98% to 100% homologous with a sequence identity of any of the variants disclosed herein, including SEQ ID NOS: 1-5, 7-8.

In some embodiments, the spore-forming bacteria is Bacillus subtilis. Spores of several aerobic species are ubiquitous in nature (Nicholson WL: Roles of Bacillus endospores in the environment. Cell Mol Life Sci 2002, 59:410-416). Examples include Bacilli and Clostridia. The spore-forming bacteria can be any spore forming Bacillus including, without limitation, Bacillus thuringiensis, Bacillus cereus, Bacillus anthracis, Bacillus amyloliquefaciens , Bacillus weihenstephanensis; Geobacillus kaustophiluy and Geobacillus thermodenilrificans . In a specific embodiment, Bacillus subtilis can be used as the spore- forming bacteria. It has been shown that ingested spores of B. subtilis safely transit the stomach, germinate and proliferate in the upper part of the intestine (Casula G, Cutting SM: Bacillus probiotics: spore germination in the gastrointestinal tract. Appl Environ Microbiol 2002, 68:2344-2352). In the lower part of the intestine the cells sporulate again, thus performing an entire life cycle in the animal gastro-intestinal tract (GIT) (Cutting SM: Bacillus probiotics. Food Microbiol. 2011, 28: 214-220) In the GIT, B. subtilis interacts with intestinal epithelial and immune cells, contributes to the normal development of the gut- associated lymphoid tissue (GALT) and protects the host from enteropathogens (Ricca, E., Baccigalupi, L., Cangiano, G. et al. Mucosal vaccine delivery by non-recombinant spores of Bacillus subtilis . Microb Cell Fact 13, 115 (2014), disclosed herein for its teaching concerning using B. subtilis spores to form vaccines).

The spore coat protein can be isolated. Methods of isolating spore coat protein are known in the art. An example of a spore coat protein that can be used in the present invention is found in SEQ ID NO: 6 (spore coat protein derived from B. subtilis). One of skill in the art will understand that this spore coat protein can be modified in multiple ways to be more efficacious in the vaccines disclosed herein. The spore coat protein used with the compositions and methods disclosed herein can be 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homologous with SEQ ID NO: 6. In some embodiments, the spore coat protein used with the compositions and methods disclosed herein can be from 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100% homologous with SEQ ID NO: 6. In further embodiments, the spore coat protein used with the compositions and methods disclosed herein can be 60% or greater, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, or 95% or greater homologous with SEQ ID NO: 6. In certain embodiments, the spore coat protein used with the compositions and methods disclosed herein can be from 80% to 82%, 82% to 84%, 84% to 86%, 86% to 88%, 88% to 90%, 90% to 92%, 92% to 94%, 94% to 96%, 96% to 98%, or 98% to 100% homologous with SEQ ID NO:6.

Foreign antigens have been expressed in B. subtilis on the surface and inside vegetative cells and on the surfaces of spores. Both vegetative cells and spores have been used as delivery vectors (Paccez et al. 2007. Evaluation of different promoter sequences and antigen sorting signals on the immunogenicity of Bacillus subtilis vaccine vehicles. Vaccine 25:4671-4680), but for oral immunization, several rounds of high doses of spores (>10¹⁰ spores per dose) have been found to be necessary (Acheson et al. 1997. Heat-stable spore- based vaccines: surface expression of invasin-cell wall fusion proteins in Bacillus subtilis , p. 179-184; Due et al. 2003. Bacterial spores as vaccine vehicles. Infect. Immun. 71:2810-2818; Oggioni et al. Bacillus spores for vaccine delivery. Vaccine 21(Suppl. 2):S96-S101; Lee et al. Development of a Bacillus subtilis-based rotavirus vaccine. Clin Vaccine Immunol. 2010; 17(11): 1647- 1655).

Any immunogenic fragment of SARS-CoV-2 can be used herein. For example, the spike protein can be used. In some embodiments, the antigenic fragment of SARS-CoV-2 is a spike protein.

The Spike protein is a large type 1 transmembrane protein which assembles itself in a trimeric structure to form a crown like appearance. The ectodomain of the spike protein of all coronaviruses are similar in organization, containing a N terminal (amino acid terminal) subunit (S-l) and a C terminal (carboxy terminal) subunit (S-2). The subunit S-l infects human respiratory epithelial cell through interaction with human ACE 2 receptor and the S- 2 subunit fuses with the host cell membrane. The spike S-l protein has been established as a key target for potential vaccine development as it has been determined to be highly immunogenic.

The spike protein used in the composition disclosed herein need not be 100% identical to the spike proteins disclosed herein. For example, the spike protein can be modified in any number of ways. “Identity” refers to a relationship between the sequences of two or more polypeptides or polynucleotides, as determined by comparing the sequences. Identity also means the degree of sequence relatedness between two sequences as determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (e.g., “algorithms”). Identity of related peptides can be readily calculated by known methods.

“Percent (%) identity” as it applies to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in the candidate amino acid or nucleic acid sequence that are identical with the residues in the amino acid sequence or nucleic acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for the alignment are well known in the art. Identity depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation. Generally, variants of a particular polynucleotide or polypeptide have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. In some embodiments, the variants of a particular polynucleotide or polypeptide can have from 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to a particular reference polynucleotide or polypeptide. In further embodiments, the variants of a particular polynucleotide or polypeptide can have 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more sequence identity to a particular reference polynucleotide or polypeptide. In certain embodiments, the variants of a particular polynucleotide or polypeptide can have from 80% to 82%, 82% to 84%, 84% to 86%, 86% to 88%, 88% to 90%, 90% to 92%, 92% to 94%, 94% to 96%, 96% to 98%, or 98% to 100% sequence identity to a particular reference polynucleotide or polypeptide. Such tools for alignment include those of the BLAST suite (Stephen F. Altschul, etal. (1997). “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs,” Nucleic Acids Res. 25:3389-3402). Another popular local alignment technique is based on the Smith-Waterman algorithm (Smith, T. F. & Waterman, M. S. (1981) “Identification of common molecular subsequences.” J. Mol. Biol. 147:195-197). A general global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, S. B. & Wunsch, C. D. (1970) “A general method applicable to the search for similarities in the amino acid sequences of two proteins.” J. Mol. Biol. 48:443-453). More recently, a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) was developed that purportedly produces global alignment of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm.

In some embodiments, the SARS-CoV-2 spike protein comprises SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

The spike protein used in the composition herein can comprise at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to SEQ ID NOS: 1-5, 7-8. In some embodiments, the spike protein used in the composition herein can comprise from 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to SEQ ID NOS: 1-5, 7-8. In further embodiments, the spike protein used in the composition herein can comprise 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more sequence identity to SEQ ID NOS: 1-5, 7-8. In certain embodiments, the spike protein used in the composition herein can comprise from 80% to 82%, 82% to 84%, 84% to 86%, 86% to 88%, 88% to 90%, 90% to 92%, 92% to 94%, 94% to 96%, 96% to 98%, or 98% to 100% sequence identity to SEQ ID NOS: 1-5, 7-8.

In some embodiments, the antigenic fragment of SARS-CoV-2 is covalently immobilized to the spore coat protein. In further embodiments, the conjugation is at an N- terminus of the spore coat protein. In certain embodiments, the conjugation is at a C-terminus of the spore coat protein. In specific embodiments, the spore coat protein is conjugated to the antigenic fragment of SARS-CoV-2 via a linker. In some embodiments, the linker is a peptide sequence.

In one embodiment, the spike protein is linked to the spore coat protein via a linker, which can be a peptide sequence. The fusion can be through a toxin-based short N-terminal peptide, which precedes the mature N-terminus of the spore coat protein. In another embodiment, fusion can be through the C-terminal peptide of the spore coat protein.

In another embodiment, the peptide linker sequence comprises a substrate sequence for an enzyme, wherein the enzyme may, for example, be a protease. A protease may include, but is not limited to, proteases in classes such as aspartic proteases, metalloproteases, cysteine proteases, serine proteases, and threonine proteases. In yet another embodiment, the linker is a dimeric linker, which may comprise a covalent association between two binding partners, such as a covalent association provided by a disulfide bond. In another embodiment the dimeric linker may comprise a non-covalent association between two partners, such as, for example, between a pair of leucine zipper peptides.

In some embodiments, the Bacillus subtilis spore coat protein comprises SEQ ID NO: 4. In further embodiments, the functional fragment thereof is 90% or more identical to SEQ ID NO: 4.

Further disclosed herein is a genetically engineered spore-forming bacteria which has been modified to express an antigenic fragment of SARS-CoV-2. In some embodiments, recombinant spore forming bacteria has been genetically engineered to express the antigenic fragment of SARS-CoV-2. Genetically-Engineered Spore Forming Bacteria, Nucleic Acids, and Cells Capable of Expressing the Nucleic Acid

In some embodiments, the genetically engineered spore-forming bacteria is Bacillus subtilis. In further embodiments, the antigenic fragment of SARS-CoV-2 is a spike protein. In certain embodiments, the SARS-CoV-2 spike protein comprises SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

Further disclosed herein is a nucleic acid encoding the compositions disclosed herein. Also disclosed are cells which are capable of expressing the nucleic acid as disclosed herein.

Vaccines

Also disclosed is a vaccine comprising the composition disclosed herein.

In some embodiments, the vaccine further comprises one or more adjuvants. In further embodiments, the vaccine is formulated for transmucosal or intranasal delivery. In certain embodiments, the vaccine is formulated for oral delivery.

In various embodiments of the invention herein, oral and intranasal inoculation using the compositions disclosed herein are contemplated. These vaccines can have a long shelf life at elevated temperatures when stored in the dry state. While intranasal administration was demonstrated to be surprisingly effective in examples herein, the antigen associated with spores leads to a vaccine that is administered in any of a variety of routes.

The vaccine can comprise an immunogenic fragment of SARS-CoV-2 from any of the variants thereof. Examples include, but are not limited to, the “wild type,” or originally discovered SARS-CoV-2 (SEQ ID NO: 5), the Indian variants (B.1.617-1, SEQ ID NO: 1, and B.1.617-2, SEQ ID NO: 2) the South African variant (B.1.351, SEQ ID NO: 3) and the Japanese/Brazilian variant (P.1, SEQ ID NO: 4). There are many other variants which are known, or will be known, which are also contemplated herein. Also disclosed are modifications to the spike protein, such as making it more soluble or more or less antigenic. Both polynucleotide and polypeptide molecules can be physically derived from a SARS- CoV-2 virus or produced recombinantly or synthetically, for example, based on known sequences. In some embodiments, the immunogenic fragment of SARS-CoV-2 can have a sequence identity 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homologous with a sequence identity of any of the variants disclosed herein, including SEQ ID NOS: 1-5, 7-8. In some embodiments, the immunogenic fragment of SARS-CoV-2 can have a sequence identity from 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100% homologous with the sequence identity of any of the variants disclosed herein, including SEQ ID NOS: 1-5, 7-8. In further embodiments, the immunogenic fragment of SARS-CoV-2 can have a sequence identity 60% or greater, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, or 95% or greater homologous with a sequence identity of any of the variants disclosed herein, including SEQ ID NOS: 1-5, 7-8. In certain embodiments, the immunogenic fragment of SARS-CoV-2 can have a sequence identity from 80% to 82%, 82% to 84%, 84% to 86%, 86% to 88%, 88% to 90%, 90% to 92%, 92% to 94%, 94% to 96%, 96% to 98%, or 98% to 100% homologous with a sequence identity of any of the variants disclosed herein, including SEQ ID NOS: 1-5, 7-8.

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active agent(s), the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active agent is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonitic clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active agent(s) may be admixed with at least one inert diluent such as sucrose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active agent(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of an inventive pharmaceutical composition include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The active agent is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. For example, ocular or cutaneous infections may be treated with aqueous drops, a mist, an emulsion, or a cream. Administration may be therapeutic or it may be prophylactic. Prophylactic formulations may be present or applied to the site of entry of potential disease organisms, such as contact lenses, contact lens cleaning and rinsing solutions, containers for contact lens storage or transport, devices for contact lens handling, eye drops, surgical irrigation solutions, ear drops, eye patches, and cosmetics for the eye area, including creams, lotions, mascara, eyeliner, and eyeshadow. The invention includes products which contain the compositions having the spores (e.g., gauze bandages or strips), and methods of making or using such devices or products. These devices may be coated with, impregnated with, bonded to or otherwise treated with a vaccine composition.

The ointments, pastes, creams, and gels may contain, in addition to an active agent of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonitic clays, silicic acid, talc, zinc oxide, or mixtures thereof. Powders and sprays can contain, in addition to the agents of this invention, excipients such as talc, silicic acid, aluminum hydroxide, calcium silicates, polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chi orofluorohy drocarb ons .

Transdermal patches have the added advantage of providing controlled delivery of the active ingredients to the body. Such dosage forms can be made by suspending spores in the matrix applied to the patches, or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the antigenic peptide released from spores into the compound, for passage across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

Method of Immunizing

In some embodiments, the spore forming bacteria is Bacillus subtilis. In further embodiments, the vaccine further comprises one or more adjuvants. In certain embodiments, the vaccine is formulated for oral delivery. In specific embodiments, the vaccine is formulated for transmucosal or intranasal use. In some embodiments, the vaccine is administered to the subject more than once. In further embodiments, the antigenic fragment of SARS-CoV-2 is a spike protein.

In some embodiments, the SARS-CoV-2 spike protein comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. The spike protein used in the method herein can comprise at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to SEQ ID NOS: 1-5, 7-8. In some embodiments, the spike protein used in the method herein can comprise from 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to SEQ ID NOS: 1-5, 7-8. In further embodiments, the spike protein used in the method herein can comprise 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more sequence identity to SEQ ID NOS: 1-5, 7-8. In certain embodiments, the spike protein used in the method herein can comprise from 80% to 82%, 82% to 84%, 84% to 86%, 86% to 88%, 88% to 90%, 90% to 92%, 92% to 94%, 94% to

96%, 96% to 98%, or 98% to 100% sequence identity to SEQ ID NOS: 1-5, 7-8.

In some embodiments, the Bacillus subtilis spore coat protein comprises SEQ ID NO: 6. In further embodiments, the functional fragment thereof is 90% or more identical to SEQ ID NO: 6. In some embodiments, the functional fragment thereof can comprise at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to SEQ ID NO: 6. In further embodiments, the functional fragment thereof can comprise from 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to

85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to SEQ ID NO: 6. In certain embodiments, the functional fragment thereof can comprise 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more sequence identity to SEQ ID NO: 6. In specific embodiments, the functional fragment thereof can comprise from 80% to 82%, 82% to 84%, 84% to 86%, 86% to 88%, 88% to 90%, 90% to 92%, 92% to 94%, 94% to 96%, 96% to 98%, or 98% to 100% sequence identity to SEQ ID NO: 6.

Method of Making a Vaccine

Also disclosed herein is a method of making a vaccine for SARS-CoV-2, the method comprising isolating a spore coat protein or a functional fragment thereof, wherein said spore coat is from a spore-forming bacteria, the method further comprising conjugating an antigenic fragment of SARS-CoV-2 to the spore coat protein. In some embodiments, the spore forming bacteria is Bacillus subtilis. In further embodiments, the antigenic fragment of SARS-CoV-2 is a spike protein. In certain embodiments, the SARS-CoV-2 protein comprises SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. The spike protein used in the method herein can comprise at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to SEQ ID NOS: 1- 5, 7-8. In some embodiments, the spike protein used in the method herein can comprise from 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to SEQ ID NOS: 1-5, 7-8. In further embodiments, the spike protein used in the method herein can comprise 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more sequence identity to SEQ ID NOS: 1-5, 7-8. In certain embodiments, the spike protein used in the method herein can comprise from 80% to 82%, 82% to 84%, 84% to 86%, 86% to 88%, 88% to 90%, 90% to 92%, 92% to 94%, 94% to 96%, 96% to 98%, or 98% to 100% sequence identity to SEQ ID NOS: 1-5, 7-8.

In some embodiments, the antigenic fragment of SARS-CoV-2 is covalently immobilized to the spore coat protein. In further embodiments, the conjugation is at an N- terminus of the spore coat protein. In certain embodiments, the conjugation is at a C-terminus of the spore coat protein. In specific embodiments, the spore coat protein is conjugated to the antigenic fragment of SARS-CoV-2 via a linker. In some embodiments, the linker is a peptide sequence. In further embodiments, the Bacillus subtilis spore coat protein comprises SEQ ID NO: 4. In certain embodiments, the functional fragment thereof is 90% or more identical to SEQ ID NO: 4. In some embodiments, the functional fragment thereof can comprise at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to SEQ ID NO: 4. In further embodiments, the functional fragment thereof can comprise from 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to SEQ ID NO: 4. In certain embodiments, the functional fragment thereof can comprise 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more sequence identity to SEQ ID NO: 4. In specific embodiments, the functional fragment thereof can comprise from 80% to 82%, 82% to 84%, 84% to 86%, 86% to 88%, 88% to 90%, 90% to 92%, 92% to 94%, 94% to 96%, 96% to 98%, or 98% to 100% sequence identity to SEQ ID NO: 4.

Also disclosed are methods of making the compositions disclosed herein.

A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below.

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.

Example 1: Orally delivered SARS-CoV-2 vaccine for B.1.617 (Indian variant), B.1.351 (South Africa) and P.l (Brazilian and Japanese) variants based on B. subtilis spore expressing Spike protein

Disclosed herein is an oral COVID-19 vaccine for novel B.1.351 (South Africa) and P.l (Brazilian and Japanese), andB.1.617 (Indian or Delta) variants in the recombinant spore- based protein coated platform on using B. subtilis spores. The candidate sequences from the S spike of SARS-CoV-2 were added to the B. subtilis to stimuli T and B cells in the GI system and humoral and cellular immunity were therefore induced against mutant variants of COVID-19. The sequence was designed for protective immunity for B.1.617 (Indian, Delta and Delta-Plus), B.1.351 (South Africa) and P.l (Brazilian and Japanese) variants as shown here.

To develop an optimal sequence, the wild type of full-length spike of SARS-CoV-2 were selected and mutations for each variant were placed in the sequence. Since two variants differ in a few basepairs, Spike B.1.351 (501 Y. V2 South Africa) protein was first synthesized. In the next step, this gene is mutagenized to produce P.l sequence. Indian Sequence is separately developed.

The 501Y.V2 variant, also known as 20H/501Y.V2 (formerly 20C/501Y.V2), B.1.351 lineage and colloquially known as South African COVID-19 variant. Lineage P.l, also known as 20J/501Y.V3, Variant of Concern 202101/02 (VOC-202101/02) (Public Health England 2021) or colloquially known as the Brazil(ian) variant.

B.l.617-1 (Indian variant) PANGO on 1 April 2021 (SEQ ID NO: 1): MF VFLVLLPL V S SQC VNLTTRT QLPP AYTN SFTRGVYYPDK VFRS S VLHSTQDLFLP FF SN VT WFH AIH V S GTN GTKRFDNP VLPFNDGVYF AS TEK SNIIRGWIF GTTLD SKT Q SLLI VNN ATN V VIK V CEF QF CNDPFLD V YYHKNNK S WME SEFRV Y S S ANN C TFE YVSQPFLMDLEGKQGNFKNLREF VFKNIDGYFKIY SKHTPINLVRDLPQGF S ALEPL VDLPIGINITRFQTLLALHRS YLTPGD S S SGWT AGAAAYYV GYLQPRTFLLKYNEN GTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGE VFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNV Y AD SF VIRGDEVRQIAPGQTGNIAD YNYKLPDDFTGC VIAWN SNNLD SK VGGNYN YRYRLFRK SNLKPFERDIS TEI Y Q AGS TPCN GVQGFN C YFPLQ S YGF QPTN GV GY QP YRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQ QFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEV P VAIHADQLTPTWRVYSTGSNVF QTRAGCLIGAEHVNN S YECDIPIGAGIC AS Y QTQ TNSRRRARSVASQSIIAYTMSLGVENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSV DCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTP PIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLIC AQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFN GIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLV KQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASA NL A ATKM SEC VLGQ SKRVDF C GKGYHLM SFPQ S APHGVVFLH VT YVP AQEKNF TT APAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVN NTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAK NLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCC SCGSCCKFDEDDSEPVLKGVKLHYT

B.l.617-2 (Indian variant) PANGO on 1 April 2021 (SEQ ID NO: 2) MF VFLVLLPL V S SQC VNLTTRT QLPP AYTN SFTRGVYYPDKVFRS S VLHSTQDLFLP FF SN VT WFH AIH V S GTN GTKRFDNP VLPFNDGV YF AS TEK SNIIRGWIF GTTLD SKT Q SLLI VNN ATN V VIK V CEF QF CNDPFLD V YYHKNNK S WME SEFRV Y S S ANN C TFE YVSQPFLMDLEGKQGNFKNLREF VFKNIDGYFKIY SKHTPINLVRDLPQGF S ALEPL VDLPIGINITRFQTLLALHRS YLTPGD S S SGWT AGAAAYYV GYLQPRTFLLKYNEN GTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGE VFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNV

Y AD SF VIRGDEVRQIAPGQTGNIAD YNYKLPDDFTGC VIAWN SNNLD SK VGGNYN YRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVQGFNCYFPLQSYGFQPTNGVGYQP YRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQ QFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEV P VAIHADQLTPTWRVYSTGSNVF QTRAGCLIGAEHVNN S YECDIPIGAGIC AS Y QTQ TNSRRRARSVASQSIIAYTMSLGVENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSV DCTMYICGD STEC SNLLLQ YGSFCTQLNRALTGIAVEQDKNTQEVF AQ VKQIYKTP PIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLIC AQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFN GIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLV

KQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASA

NL A ATKM SEC VLGQ SKRVDF C GKGYHLM SFPQ S APHGVVFLH VT YVP AQEKNF TT

APAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVN

NTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAK

NLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCC

SCGSCCKFDEDDSEPVLKGVKLHYT

B.1.351 (20H/501Y.V2) South Africa Oct 2020 (SEQ ID NO: 3)

MF VFLVLLPL V S SQC VNLTTRT QLPP AYTN SFTRGVYYPDKVFRS S VLHSTQDLFLP FF SN VT WFH AIH V S GTN GTKRFDNP VLPFNDGV YF AS TEK SNIIRGWIF GTTLD SKT QSLLIVNNATNVVIKVCEF QF CNDPFLGVYYHKNNKSWMESEFRVY S S ANNCTFE YVSQPFLMDLEGKQGNFKNLREF VFKNIDGYFKIY SKHTPINLVRDLPQGF S ALEPL VDLPIGINITRFQTLL ALHRS YLTPGD S S SGWT AGAAAYYV GYLQPRTFLLK YNEN GTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGE VFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNV Y AD SF VIRGDEVRQI APGQTGNIAD YNYKLPDDFTGC VIAWN SNNLD SK VGGNYN YL YRLFRK SNLKPFERDI STEI Y Q AGS TPCN GVKGFNC YFPLQ S Y GF QPT YGV GY QP YRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQ QFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEV P VAIHADQLTPTWRVYSTGSNVF QTRAGCLIGAEHVNN S YECDIPIGAGIC AS Y QTQ TNSPRRARSVASQSIIAYTMSLGVENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSV DCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTP PIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLIC AQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFN GIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLV KQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASA NLA ATKM SEC VLGQ SKRVDF C GKGYHLM SFPQ S APHGVVFLH VT YVP AQEKNF TT APAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVN NTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAK NLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCC SCGSCCKFDEDDSEPVLKGVKLHYT

P.l (20J/501Y.V3) Brazil and Japan Jan 2021 (SEQ ID NO: 4) MF VFLVLLPL V S SQCVNFTTRTQLPS AYTN SFTRGVYYPDKVFRS SVLHSTQDLFLP FF SN VT WFH AIH V S GTN GTKRFDNP VLPFNDGVYF AS TEK SNIIRGWIF GTTLD SKT QSLLIVNNATNVVIKVCEF QF CNYPFLGVYYFKNNKSWMESEFRVY S S ANNCTFE YVSQPFLMDLEGKQGNFKNLSEF VFKNIDGYFKIY SKHTPINLVRDLPQGF S ALEPL VDLPIGFNilTRFQTLL ALHRS YLTPGD S S SGWT AGAAAYYV GYLQPRTFLLK YNEN GTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGE VFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNV

Y AD SF VIRGDE VRQI APGQTGTI AD YN YKLPDDF T GC VI AWN SNNLD SK V GGNYN YL YRLFRK SNLKPFERDI STEI Y Q AGS TPCN GVKGFNC YFPLQ SY GF QPT YGV GY QP YRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQ QFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEV P VAIHADQLTPTWRVYSTGSNVF QTRAGCLIGAEYVNN S YECDIPIGAGIC AS Y QTQ TNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSV DCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTP PIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLIC AQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFN GIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLV KQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASA NLAAIKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTT APAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVN NTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAK NLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCC SCGSCCKFDEDDSEPVLKGVKLHYT

>pdb|7DXl|A Chain A, Wild type Spike glycoprotein, (SEQ ID NO: 5)

MF VFLVLLPL V S SQC VNLTTRT QLPP AYTN SFTRGVYYPDKVFRS S VLHSTQDLFLP FF SN VT WFH AIH V S GTN GTKRFDNP VLPFNDGVYF AS TEK SNIIRGWIF GTTLD SKT QSLLIVNNATNVVIKVCEF QF CNDPFLGVYYHKNNKSWMESEFRVY S S ANNCTFE YVSQPFLMDLEGKQGNFKNLREF VFKNIDGYFKIY SKHTPINLVRDLPQGF S ALEPL VDLPIGINITRFQTLLALHRS YLTPGD S S SGWT AGAAAYYV GYLQPRTFLLKYNEN GTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGE VFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNV

Y AD SF VIRGDEVRQI APGQTGKIAD YNYKLPDDFTGC VIAWN SNNLD SK VGGNYN YL YRLFRK SNLKPFERDI STEI Y Q AGS TPCN GVEGFN C YFPLQ SYGF QPTN GV GY QP YRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQ QFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEV

P VAIHADQLTPTWRVYSTGSNVF QTRAGCLIGAEHVNN S YECDIPIGAGIC AS Y QTQ

TNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSV

DCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTP

PIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLIC

AQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFN

GIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLV

KQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASA

NL A ATKM SEC VLGQ SKRVDF C GKGYHLM SFPQ S APHGVVFLH VT YVP AQEKNF TT

APAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVN

NTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAK

NLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCC

SCGSCCKFDEDDSEPVLKGVKLHYTLEDYKDDDDK

SPORE COAT PROTEIN from B. SUBTILIS. GenBank: KOS72492.1, (SEQ ID

NO: 6)

MESRPYSWVA LDPDCDHPLE HKEKEKEERK CNCDICCNNN GF GNDNNAFI DQDLAQANLN KQVSDETIII RDSCDINVSS TDVQAVTSIV TALNAAVLTV ALTSIADGVI AELVAQDLLQ LTANKQVNRQ KLLIECSRGV NVTTVDADIA TLVSTATNVL IAVLVITLVL

All sequences are expressed in human cells and then were subcloned into pcDNA3.1(+). CotZ was used as a stable anchor protein to link the purified peptide to the spore surface display in B. subtilis as previously described (Hinc 2013).

The spike proteins attached to the purified spores of B. subtilis (Fig. 1). For immobilization of spike on the spore surface by covalent binding, initially, the free carboxyl groups on the spore surface activate with 1 -ethyl-3 -(3 -dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NITS), then the reaction between activated carboxyl groups of the spore and amino groups of the enzyme performs and amide bonds were formed between them (Fig. 2).

Methods

Sequence production: The sequences were manufactured by Proteogenix LLC, Strasbourg France.

Preparation of spores: Sporulation was made in Difco Sporulation Medium (DSM) by the exhaustion method as described (Leighton and Doi 1971; Nicholson and Setlow; 1990). Sporulating cultures were harvested 24 hours after the initiation of sporulation, treated with lysozyme to break residual sporangial cells, then washed in 1 M NaCl, 1 M KC1, and two times with water. Phenylmethylsulfonylfluoride (1 mM) is added to inhibit proteolysis. Spores are treated at 65°C for 1 hour to kill residual cells after the final wash. The number of spores obtained after purification were calculated by direct counting with a Biirker chamber under an optical microscope. By this method, approximately 10¹¹ spores per 1 ml of DSM medium were produced.

Spore binding and Western blot analysis: 1 -ethyl-3 -(3 -dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS) cross-linkers, were employed to immobilization of spike on the surface of B. subtilis spores by covalent bindings as described by (Hermanson 2008) (Fig. 2). Spore coat proteins were extracted from suspensions of spores at high density (>1 ^c 10¹⁰ spores per ml) using a sodium dodecyl sulfate (SDS)-dithiothreitol extraction buffer as described in detail elsewhere (Nicholson and Setlow 1990). Extracted proteins were subjected to 12% SDS-polyacrylamide gel electrophoresis (PAGE) and visualized by Coomassie brilliant blue G-250 staining. The proteins were transferred onto a cellulose nitrate membrane after SDS-PAGE. The membrane was incubated with rat anti-TP20.8 serum, then identified by horseradish peroxidase (HRP)- conjugated rabbit anti-rat antibody (Sigma) and visualized by diaminobenzidine tetrahydrochloride substrate solution.

Immunization of rats and sample collection: Groups of ten Sprague-Dawley rats (male, 8 weeks) were immunized by the oral route with suspensions of either spores expressing CotC-TP20.8 or control Cot C spores. A naive, nonimmunized control group was enrolled. Rats were administrated orally with 1.0 ^c 10¹⁰ spores in a volume of 0.15 ml by intragastric lavage on days 0, 1, 2, 16, 17, 18, 33, 34, and 35. Serum and feces samples were collected on days -1, 15, 32, and 50.

The fecal samples were treated following the procedure introduced by Robison et al. (1997). Fecal pellet (0.1 g) is suspended in phosphate-buffered saline (PBS) with bovine serum albumin (BSA; 1%) and 1 mM phenylmethylsulfonyl fluoride, incubated at 4°C overnight, and then centrifuged; the supernatant was stored at -20°C before enzyme-linked immunosorbent assay (ELISA). Serum samples were stored for IgG against S protein measurements. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference. Those skilled in the art will appreciate that numerous changes and modifications can be made to the preferred embodiments of the invention and that such changes and modifications can be made without departing from the spirit of the invention. It is, therefore, intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the invention.

References

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Claims

CLAIMS What is claimed is:

1. A composition comprising an isolated spore coat protein, or a functional fragment thereof, wherein the spore coat protein is from a spore-forming bacteria conjugated to an antigenic fragment of SARS-CoV-2.

2. The composition of claim 1, wherein the spore-forming bacteria is Bacillus subtilis.

3. The composition of claim 1 or 2, wherein the antigenic fragment of SARS-CoV-2 is a spike protein.

4. The composition of claim 3, wherein the SARS-CoV-2 spike protein comprises SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

5. The composition of any one of claims 1-4, wherein the antigenic fragment of SARS- CoV-2 is covalently immobilized to the spore coat protein.

6. The composition of any one of claims 1 -5, wherein the conjugation is at an N-terminus of the spore coat protein.

7. The composition of any one of claims 1-5, wherein the conjugation is at a C-terminus of the spore coat protein.

8. The composition of any one of claims 1-7, wherein the spore coat protein is conjugated to the antigenic fragment of SARS-CoV-2 via a linker.

9. The composition of claim 8, wherein the linker is a peptide sequence.

10. The composition of claim 2, wherein the Bacillus subtilis spore coat protein comprises SEQ ID NO: 4.

11. The composition of claim 10, wherein the functional fragment thereof is 90% or more identical to SEQ ID NO: 4.

12. A nucleic acid encoding the composition of any one of claims 1-11.

13. A cell capable of expressing the nucleic acid of claim 12.

14. A genetically engineered spore-forming bacteria which has been modified to express an antigenic fragment of SARS-CoV-2.

15. The genetically engineered spore-forming bacteria of claim 14, wherein the spore forming bacteria is Bacillus subtilis.

16. The genetically engineered Bacillus subtilis bacteria of claim 14 or 15, wherein the antigenic fragment of SARS-CoV-2 is a spike protein.

17. The genetically engineered Bacillus subtilis bacteria of claim 16, wherein the SARS- CoV-2 spike protein comprises SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

18. A vaccine comprising the composition of claim 1.

19. The vaccine of claim 18, wherein the vaccine further comprises one or more adjuvants.

20. The vaccine of claim 18 or 19, wherein the vaccine is formulated for transmucosal or intranasal delivery.

21. The vaccine of any one of claims 18-20, wherein the vaccine is formulated for oral delivery.

22. A method of immunizing a subject against SARS-CoV-2 infection, the method comprising administering to the subject a vaccine, wherein the vaccine comprises an isolated spore coat protein or a functional fragment thereof, wherein said spore coat protein is from a spore-forming bacteria, wherein the spore coat protein is conjugated to an antigenic fragment of SARS-CoV-2.

23. The method of claim 22, wherein the spore forming bacteria is Bacillus subtilis.

24. The method of claim 22 or 23, wherein the vaccine further comprises one or more adjuvants.

25. The method of any one of claims 22-24, wherein the vaccine is formulated for oral delivery.

26. The method of claim 25, wherein the vaccine is formulated for transmucosal or intranasal use.

27. The method of any one of claims 22-26, wherein the vaccine is administered to the subject more than once.

28. The method of any one of claims 22-27, wherein the antigenic fragment of SARS- CoV-2 is a spike protein.

29. The method of claim 28, wherein the SARS-CoV-2 spike protein comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

30. The method of claim 23, wherein the Bacillus subtilis spore coat protein comprises SEQ ID NO: 6.

31. The method of claim 30, wherein the functional fragment thereof is 90% or more identical to SEQ ID NO: 6.

32. A method of making a vaccine for SARS-CoV-2, the method comprising isolating a spore coat protein or a functional fragment thereof, wherein said spore coat protein is from a spore-forming bacteria, the method further comprising conjugating an antigenic fragment of SARS-CoV-2 to the spore coat protein.

33. The method of claim 32, wherein the spore forming bacteria is Bacillus subtilis.

34. The method of claim 32 or 33, wherein the antigenic fragment of SARS-CoV-2 is a spike protein.

35. The method of claim 34, wherein the SARS-CoV-2 spike protein comprises SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

36. The method of any one of claims 32-35, wherein the antigenic fragment of SARS- CoV-2 is covalently immobilized to the spore coat protein.

37. The method of any one of claims 32-36, wherein the conjugation is at an N-terminus of the spore coat protein.

38. The method of any one of claims 32-36, wherein the conjugation is at a C-terminus of the spore coat protein.

39. The method of any one of claims 32-38, wherein the spore coat protein is conjugated to the antigenic fragment of SARS-CoV-2 via a linker.

40. The method of claim 39, wherein the linker is a peptide sequence.

41. The method of claim 33, wherein the Bacillus subtilis spore coat protein comprises SEQ ID NO: 4.

42. The method of claim 41, wherein the functional fragment thereof is 90% or more identical to SEQ ID NO: 4.